In 1975 Kohler and Milstein introduced a procedure for the production of monoclonal antibodies (mAbs) using hybrid cells (hybridomas) which allows the production of almost unlimited quantities of antibodies of precise and reproducible specificity. Conventional antisera, produced by immunizing animals with tumor cells or other antigens, contain a myriad of different antibodies differing in their specificity and properties, whereas hybridomas produce a single antibody with uniform characteristics. The Kohler-Milstein procedure entails the fusion of spleen cells from an immunized animal with an immortal myeloma cell line. From the fused cells (hybridomas), clones are selected that produce antibody of the desired specificity. Each clone continues to produce only that one antibody. As hybridoma cells can be cultured indefinitely (or stored frozen in liquid nitrogen), a constant supply of antibody is assured.
Antibodies are proteinaceous molecules that have the ability to combine with and recognize other molecules, known as antigens. Monoclonal antibodies are no different from other antibodies except that they are very uniform in their properties and are believed to recognize only one antigen or a portion of an antigen known as a determinant.
In the case of cells, the determinant recognized is an antigen on or in the cell which reacts with the antibody. It is through these cell antigens that a particular antibody recognizes, i.e. reacts with, a particular kind of cell. Thus the cell antigens are markers by which the cell is identified.
These antigenic markers may be used to observe the normal process of cell differentiation and some are sensitive enough to locate differences and abnormalities within a given cell system. The process of differentiation is accompanied by changes in the cell surface antigenic phenotype, and antigens that distinguish cells belonging to distinct differentiation lineages or distinguish cells at different phases in the same differentiation lineage may be observed if the correct antibody is available. Tumor antigens may also be detected. Initial recognition of differentiation antigens came about through analysis of surface antigens of T-cell leukemias of the mouse and the description of the TL, Thy-1, and Lyt series of antigens. (Old, Lloyd J., Cancer Research, 41, 361-375, February 1981) The analysis of these T-cell differentiation antigens was greatly simplified by the availability of normal T cells and B cells of mouse and man. (See U.S. Pat. Nos. 4,361,549-550; 4,364,932-37 and 4,363,799 concerning mAb to Human T-cell antigens).
Progress in defining surface antigens on melanocytes was made possible by the recently discovered technique of culturing melanocytes from normal skin [Eisinger, et al., Proc Nat'l. Acad. Sci. U.S.A., 79 2018 (March 1982), copending patent application Ser. No. 469,854, abandoned. ] This method provides a renewable source of proliferating cells for the analysis of melanocyte differentiation antigens.
Cell surface antigens of human malignant melanoma identified by mouse monoclonal antibodies are described as well [Dippold et al. Proc. Nat'l. Acad. Sci. U.S.A. 77, 6114-6118 (1980).] Previous work in human cancer is found in a co-pending patent application Ser. No. 297,814 Monoclonal Antibodies To Cell Surface Antigens of Human Renal Cancer.
The preparation of hybrid cell lines can be successful or not depending on such experimental factors as nature of the innoculant, cell growth conditions, hybridization conditions etc. Thus it is not always possible to predict successful hybridoma preparation of one cell line although success may have been achieved with another cell line. This is due to the difficulty of obtaining a ready source of the appropriate normal cell type as well as the vagaries of the art of monoclonal antibodies.
Functional subpopulations of human T lymphocytes were first demonstrated using heterologous anti-human T cell sera (Evans, R. L., et al. (1977) J. Exp. Med. 145:221; Evans, R. L., et al., (1978) J. Immunol. 120:1423). The introduction of somatic cell hybridization techniques for the production of monoclonal antibodies (mAb) by Kohler and Milstein (Kohler, G., and C. Milstein. (1975) Nature 256:195) has resulted in rapid development of many antibodies for the detection of unique cell surface antigens, selectively expressed on human T lymphocytes and their subpopulations. The human T lymphocyte antigens detected by such antibodies can be divided into at least three groups: (1) pan T cell antigens, which are expressed on virtually all peripheral T cells and/or thymocytes; (2) T cell antigens expressed on inducer/helper T lymphocytes; and (3) antigens expressed on cytotoxic/suppressor T lymphocytes. To date, pan T cell antigens comprise at least three different molecules with m.w. ranges of about 40,000 to 50,000 [e.g., defined by mAb 9.6 (Kamoun, M., et. al. (1981) J. Exp. Med. 153:207, and mAb Leu 5 (Howard, et al. (1981), J. Immunol. 126:2117] 67,000 to 70,000 [e.g., defined by mAb OKT 1 (Kung, P. C., et al. (1979) Science, 206:347; Reinherz, E. L., (1979) J. Immunol. 123:1312); (P. J. Martin, et. al. (1980) Immunogenetics, 11:429), mAb Leu 1 (Wang, C. Y., (1980) J. Exp. Med. 151:1539), mAb T 101 (Royston, I., et al. (1980) J. Immunol. 125:725), and mAb L17F12 (Engleman, E. G., et. al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1791); and 20,000 to 30,000 (e.g., defined by mAb OKT 3 (Kung, P. C., Supra mAb Leu 4 (Ledbetter, J. A., et al. (1981) In Monoclonal Antibodies and T Cell Hybridomas. G. J. Hammerling, and J. F. Kearney, eds. Elsevier/North Holland, New York. In press), and mAb 38.1 (Hansen, J. A., and P. J. Martin. (1981) In Workshop on Hybridomas in Cancer Diagnosis and Treatment. M. Mitchell and H. Oettgen, eds. Raven Press, New York. In press). The inducer/helper T lymphocyte subpopulation is defined by mAb OKT 4 (Kung, P. C., et. al. Supra) Reinherz, E. L., et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:4061) and mAb Leu 3a (Evans, R. L. et. al. (1981) Thymus-dependent membrane antigens in Proc. Nat. Acad. Sci. U.S.A. 78:544); (Ledbetter, J. A., et al. (1981) J. Exp. Med. 153:310); (Engleman, E. G., et. al. (1981) J. Exp. Med. 153:193). The human T lymphocyte subpopulation containing the cytotoxic/suppressor T cells are defined by other mAb, mAb OKT 5 (Reinherz, et al. (1980) J. Immunol. 124:1301), mAb OKT 8 (Thomas, Y., et. al. (1980) J. Immunol. 125:2402), and mAb Leu 2a (Evans, (1981) Supra, Ledbetter, Supra, Engleman, E. G., J. Exp. Med. Supra). Haynes et al. (Haynes, B. F., et. al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:5829; Haynes, B. F., et. al. (1980) Proc. Nat. Acad. Sci. U.S.A. 77:2914; Haynes, B. F., et. al. (1981) N. Engl. J. Med. 304:1319; Haynes, B. F. (1981) Immunol. Rev. 57:127) have described a human T lymphocyte specific antigen detected by mAb 3A1. This antibody detects a cell surface antigen with m.w. of 40,000, which is expressed on T helper cells involved in pokeweed mitogen- (PWM) driven in vitro antibody production and on the concanavalin A- (Con A) induced cells that cause in vitro suppression of B cell Ig synthesis.